Engineering and functional immobilization of opioid receptors

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCR

Characterization of carbon fibrous material from platanus achenes as platinum catalysts support

Carbon materials with developed porosity are usually used as supports for platinum catalysts. Physico-chemical characteristics of the support influence the properties of platinum deposited and its catalytic activity. In our studies, we deposited platinum on carbon fibrous like materials obtained from platanus seeds - achenes. The precursor was chemically activated with different reagents: NaOH, pyrogallol, and H2O2, before the carbonization process. Platinum was deposited on all substrates to study the influence of the substrate properties on the activity of the catalyst. Carbon materials were characterized by nitrogen adsorption/desorption isotherms measurements, X-ray diffraction, and scanning electron microscopy. It was noticed that the adsorption characteristics of carbon support affected the structure of platinum deposits and thus their activity

Evolution of three human GPCRs for higher expression and stability

We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target-the tachykinin receptor NK(1), which is hardly expressed in E. coli and cannot be functionally solubilized-receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein

Engineering and functional immobilization of opioid receptors

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs

Characterization analysis of activated carbon derived from the carbonization process of plane tree (Platanus orientalis) seeds

In this work, plane tree seed-based activated carbons were characterized in detail for a variety of applications. The particularly important area of application would be in the artificial photosynthesis. After carbonization process of biomass precursor at 650°C, the resulting preliminary activated carbons were activated at various temperatures. The activated carbons were characterized by oxygen functionalities (a particularly important role has ester oxygen groups) which provide a unique microstructure. The chemical compositions of as-prepared activated carbons were analyzed through Fourier transform infrared and Raman spectra as well as gas chromatography–mass spectroscopy analysis, while morphology was observed by scanning electron microscopy analysis. Applied analysis showed that detected graphite mainly becomes uniformly nanocrystalline system. The current study also explored the applicability of carbon material obtained from plane tree seed as a potential gaseous adsorbent. The characterization showed that the tested material contains both mesopores and micropores, and this should be advantageous for the gas sorption process, since mesopores may provide low-resistant pathways for the diffusion of CO2 molecules, while the micropores are the most suitable for trapping of CO2. The sorption process analysis (including adsorption/desorption isotherms behavior) shows indication that the rate-limiting step of CO2 adsorption onto activated carbon is probably governed by diffusion-controlled process, especially at temperatures below 850°C

Characterization of carbon fibrous material from platanus achenes as platinum catalysts support

Carbon materials with developed porosity are usually used as supports for platinum catalysts. Physico-chemical characteristics of the support influence the properties of platinum deposited and its catalytic activity. In our studies, we deposited platinum on carbon fibrous like materials obtained from platanus seeds - achenes. The precursor was chemically activated with different reagents: NaOH, pyrogallol, and H2O2, before the carbonization process. Platinum was deposited on all substrates to study the influence of the substrate properties on the activity of the catalyst. Carbon materials were characterized by nitrogen adsorption/desorption isotherms measurements, X-ray diffraction, and scanning electron microscopy. It was noticed that the adsorption characteristics of carbon support affected the structure of platinum deposits and thus their activity.</jats:p

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ((2)H/(15)N/(13)C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K(D) values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering